More than 75 million people in the U.S. suffer from chronic disabling pain (NIH Guide, 1998). Chronic pain in America is a large social and economic burden, with costs exceeding $50 billion annually in lost wages, lost productivity, medical expenses, and the like. Additional costs are more difficult to quantify, such as the physical and emotional impacts on a pain sufferer and their family members.
Normal pain is an important self-protection mechanism employed by the body. Upon the occurrence of harmful stimulus, the peripheral nociceptors (pain-sensing primary afferent neurons) detect and send the signal of pain through Aβ, Aδ, and C fibers to the dorsal horn of the spinal cord. The dorsal horn processes the incoming signals, and upon accumulation of signals, transmits the information to supraspinal sites that in turn dictate a response, for example, withdrawal of a foot from heat. In a normal, physiological pain response, the pain sensation resolves upon cessation of the harmful stimulus.
Chronic pain, unlike normal pain, does not abate. A number of physiological changes in the spinal cord, dorsal root ganglia (DRG), and the brain have been observed, which correspond to the state of chronic pain. The exact mechanism of the evolution of chronic pain has not been elucidated; however, central sensitization has been shown to play a role in the onset of chronic pain. C fibers are likely to be dominantly activated in most cases of chronic pain based on evidence that these fibers are predominately activated in tests employing a low rate of heating, while high-rate heating activates Aδ fibers.
Chronic neuropathic pain results from aberrant sensory processing in either the peripheral and/or the central nervous system (CNS), typically caused by an initial inflammatory, immunological, or viral episode, or by ischemic or mechanical insult to a nerve. Neuropathic pain is characterized by an altered pain perception that can manifest as allodynia, a response to a normally non-noxious stimulus (e.g., the touch of clothing becomes painful), or as hyperalgesia, a decreased threshold to noxious stimuli (e.g., warm water on burned skin).
Traditional pharmacological therapies and surgical intervention are ineffective in treating many types of pain. Therapies that do exist, such as opioids, are often ineffective in the long-term due to the development of tolerance and side effects. Therefore, there remains a great need for new, highly specific agents which, when used alone or in conjunction with existing therapies, would alleviate suffering from pain.
Protein kinase C (PKC) is a key enzyme in signal transduction involved in a variety of cellular functions, including cell growth, regulation of gene expression, and ion channel activity. The PKC family of isozymes includes at least 11 different protein kinases which can be divided into at least three subfamilies based on their homology and sensitivity to activators.
Members of the classical or cPKC subfamily, α, βI, βII and γPKC, contain four homologous domains (C1, C2, C3 and C4) inter-spaced with isozyme-unique (variable or V) regions, and require calcium, phosphatidylserine (PS), and diacylglycerol (DG) or phorbol esters for activation. The classical PKC family, α, βI, βII, and γ isozymes are found in the superficial laminae of the dorsal horn in the spinal cord.
Members of the novel or nPKC subfamily, δ,ε,η, and θPKC, lack the C2 homologous domain and do not require calcium for activation. εPKC is found in primary afferent neurons both in the dorsal root ganglia (DRG) as well as in the superficial layers of the dorsal spinal cord.
Finally, members of the atypical or αPKC subfamily, ζ and λ/αIPKC, lack both the C2 and one half of the C1 homologous domains and are insensitive to DG, phorbol esters, and calcium.
Studies on the subcellular distribution of PKC isozymes demonstrate that activation of PKC results in its redistribution in the cells (also termed translocation), such that activated PKC isozymes associate with the plasma membrane, cytoskeletal elements, nuclei, and other subcellular compartments (Saito, N. et al., Proc. Natl. Acad. Sci. USA 86:3409-3413 (1989); Papadopoulos, V. and Hall, P. F. J. Cell Biol. 108:553-567 (1989); Mochly-Rosen, D., et al., Molec. Biol. Cell (formerly Cell Reg.) 1:693-706, (1990)).
The unique cellular functions of different PKC isozymes are determined by their subcellular location. For example, activated βIPKC is found inside the nucleus, whereas activated βIIPKC is found at the perinucleus and cell periphery of cardiac myocytes (Disatnik, M. H., et al., Exp. Cell Res. 210:287-297 (1994)). The localization of different PKC isozymes to different areas of the cell in turn appears due to binding of the activated isozymes to specific anchoring molecules termed Receptors for Activated C-Kinase (RACKs). RACKs are thought to function by selectively anchoring activated PKC isozymes to their respective subcellular sites. RACKs bind only fully activated PKC and are not necessarily substrates of the enzyme. Nor is the binding to RACKs mediated via the catalytic domain of the kinase (Mochly-Rosen, D., et al., Proc. Natl. Acad. Sci. USA 88:3997-4000 (1991)). Translocation of a PKC reflects binding of the activated enzyme to RACKs anchored to the cell particulate fraction and the binding to RACKs is required for a PKC to produce its cellular responses (Mochly-Rosen, D., et al., Science 268:247-251 (1995)). Inhibition of PKC binding to RACKs in vivo inhibits PKC translocation and PKC-mediated function (Johnson, J. A., et al., J. Biol. Chem 271:24962-24966 (1996a); Ron, D., et al., Proc. Natl. Acad. Sci. USA 92:492-496 (1995); Smith, B. L. and Mochly-Rosen, D., Biochem. Biophys. Res. Commun. 188:1235-1240 (1992)).
In general, translocation of PKC is required for proper function of PKC isozymes. Peptides that mimic either the PKC-binding site on RACKs (Mochly-Rosen, D., et al., J. Biol. Chem., 226:1466-1468 (1991a); Mochly-Rosen, D., et al., supra, 1995) or the RACK binding site on PKC (Ron, et al., supra, 1995; Johnson, J. A. et al., supra, 1996a) are isozyme-specific translocation inhibitors of PKC that selectively inhibit the function of the enzyme in vivo.
Three PKC isozymes have been shown to participate in the sensation of pain (the nociception pathway): βII, γ, and ε (Igwe O. J., et al., Neuroscience 104(3):875-890 (2001); Martin W. J., et al., Neuroscience 88(4):1267-1274 (1999) Khasar S. G., et al., Neuron 24(1):253-60 (1999)). βIIPKC was found to be activated in hyperalgesia induced by peripheral inflammation with complete Freund's adjuvant (Igwe O. J., et al., Neuroscience 104(3):875-890 (2001)). Another study suggested that γPKC was activated upon injury with the same agent (Martin W. J., et al., J. Neuroscience 21(14):5321-5327 (2001)), and that γPKC deficient mice show greatly reduced hyperalgesia following an inflammatory nerve injury (Martin W. J., et al., Neuroscience 88(4):1267-1274 (1999)). εPKC deficient mice exhibit attenuated hyperalgesic responses to thermal stimulation following inflammation, suggesting that εPKC also plays an important role in nociceptor function (Khasar S. G., et al., Neuron 24(1):253-60 (1999)). Use of non-specific PKC inhibitors like calphostin in a neuropathy model (Ohsawa M., et al., Eur. J. Pharmacol., 372(3):221-8 (1999)), NPC15437 in a capsaicin model (Sluka K. A., et al., Pain, 71(2):165-178 (1997)), and chelerythrine in a formalin model (Hua X. Y., et al., Neurosci Lett., 276(2):99-102 (1999)) all showed reversal of the allodynia and/or hyperalgesia induced by the inflammatory agents.
The role of εPKC in pain perception has also been described (WO 00/01415; U.S. Pat. No. 6,376,467), and the εV1-2 peptide, a selective inhibitor of εPKC, was reported to lessen pain.
Despite such findings that PKC in general appears to play a role in nociception, few peptide sequences involved in nociception have been identified. To date, only a handful of εPKC V1 peptides have been described as therapeutically effective for the management of pain. The present invention is concerned with providing additional PKC isozyme targets and PKC isozyme/region specific peptides for the development of non-opioid based pain treatments.